Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.


Nanoflow SCX -native mass spectrometry (nMS)
Nanoflow strong cation exchange chromatography (SCX) was performed on an UltiMate RSLCnano system (Thermo Fisher Scientific, Breda, The Netherlands) equipped with a high-pressure pump with microflow selector and a loading pump (NCS-3500RS), a binary nano/capillary pump (NCP-3200RS), a thermostatted column compartment (equipped with two 10-port, two-position valves) and an autosampler.Injection loops of 1 μL (without trap) and 20 μL (with trap) were used.The autosampler was kept at 7 °C during the analysis.A QExactive-Plus Biopharma high-resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was employed.Nanospray ionization was realized with a nanospray-flex-series ion-source platform (Thermo Fisher Scientific), a Simple Link UNO (1/32, Fossiliontech, Albacete, Spain), and a nano emitter (75 mm length × 20 µm I.D., Fossiliontech) with hydrophobic coating were used.

Data analysis
The data from HPLC-UV/FLD were analyzed with the Agilent OpenLAB CDS Chemstation software (offline).The data from nanoflow SCX-nMS were analyzed with Thermo Xcalibur Qual Browser (version 4.4) and Freestyle software (version 1.7, Thermo Fisher Scientific).The UniDec program (University of Arizona, Phoenix, AZ, USA) and BioPharma Finder (version 4.0, Thermo Fisher Scientific) was used to perform deconvolution of mass spectra and obtain the molecular weight (MW). [1]Total ion chromatogram (TIC), extracted ion chromatogram (EIC), and base peak chromatogram (BPC) were smoothed using a 7-point Gaussian filter.The data presented are available at the following MassIVE Repository link: ftp://massive.ucsd.edu/v07/MSV000094068/.Deconvoluted parameters for Figure 3g and S9 are as follows: Time start: 10 to 40 min; Sliding windows (min): 1; Offset: 10; Partition in time step of: 1 min; The m/z range: 1000 to 8000; Charge range: 5 to 30; Mass range: 10,000 to 150,000 Da; Sample mass every (Da): 1; Picking range (Da): 5; Picking threshold: 0.01.

Development of salt-mediated pH gradient method
To realize the online coupling of SCX and MS, volatile salts were used.Standard mixtures of proteins (CA, BSA, Myo, and RNase-A) and monoclonal antibodies (mAbs; Pem, Cet, and Tra) were used to evaluate the developed method.20 mM or 50 mM AmAc (pH=5.0,adjusted by acetic acid) was used as mobile phase A (MPA).A series of concentrations of AmAc (140 mM to 250 mM) and pH (8.0 to 10.0, adjusted by ammonia) were investigated as mobile phase B (MPB).Proteins and mAbs mixtures were reconstituted with mobile phase A to a concentration of 1 mg/mL.The injection volume was 5 µL.The wavelengths of UV detection were set at 220, 256, and 280 nm.The excitation and emission wavelengths for FLD detection were 280 nm and 340 nm, respectively.The gradient started from 100% MPA and went to 100% MPB in 22.5 min, at a flow rate of 0.4 mL/min.To monitor the pH profile in the salt-mediated pH-gradient method, blank fractions were collected every 1 min, and the corresponding pH values were obtained using a pH meter.

Preparation of capillary SCX and trap columns
The strong cation-exchange (SCX) column, BioPro IEX SF (100 mm x 4.6 mm, 5 μm particle size, YMC, Japan), was unpacked to collect the functional resins.An empty capillary was end-sealed with a frit before use.The obtained resins were reconstituted in the packing solution, composed of 500 mM Na2SO4 and 50 mM PBS (pH 7.0), before they were flushed into the end-sealed capillary with a pump at a relatively stable pressure.After observing the capillary to be fully packed, the packing flow was maintained for at least 10 min to stabilize and compact the resin.Finally, the columns were flushed with a low concentration of volatile salts before application.

Proteins and mAbs mixtures measured by nanoflow SCX-nMS
Mobile phases composed of 50 mM AmAc (pH=5.0adjusted by acetic acid, MPA) and 250 mM AmAc (pH=8.5 adjusted by ammonia, MPB) were filtered through 0.4-µm micropore membranes before use.Laboratory-packed capillary SCX columns and traps were employed to do the measurements.Figure S4 shows the workflow of the nanoflow SCX-nMS setup (nanoSCX-nMS).To preliminarily investigate the sensitivity of nanoSCX-nMS system, a series of concentrations of BSA (from 0.1 mg/mL to 1.0 mg/mL) were tested.To evaluate the chromatographic performance, mixtures in MPA of proteins (RNase-A, Myo, CA, and BSA) and mAbs (Pem, Cet, and Tra) of 0.1 mg/mL were measured without a trap, while mixtures of 0.01 mg/mL were measured with a trap.A mobile phase of 20 mM AmAc (pH=4.5, adjusted by acetic acid) was used to load samples on the trap using the loading pump.The flow rate was set at 0.5 µL/min (for a 100-µm I.D. column) or 0.25 µL/min (for a 75-µm I.D. column).The injection volume was 1 µL or 10 µL, without or with a trap, respectively.The temperature of the autosampler was kept at 7 °C and no temperature control was installed for the column oven.The gradient ran from 0% MPB to 100% MPB in 30 min.Prior to measurements, the system was operated for at least 5 hours to be stable.
The MS instrument was operated at a spray voltage between 1.8 and 2.0 kV (depending on the conditions of the emitter), with a transfer-capillary temperature of 275 °C.The radio frequency of the S-lens was 200.The acquisition parameters were as follows.Scan mode, HRM; scan range, 1000 to 8000 m/z; in-source collisioninduced dissociation (isCID), 55 eV for protein mixtures, and 85 eV for mAbs mixtures; number of microscans, 10; resolution, 17,500; automatic-gain-control (AGC) target, 3 x 10 6 ; maximum injection time (IT), 200 ms.

Measurements of E. coli cell lysates by nanoSCX-nMS
To obtain the E. coli cell lysate, 5.13 g of E. coli cells containing overexpressed Te-ADH were suspended in 50 mL of KPi buffer with a concentration of 50 mM and pH 8 (10 mL buffer for 1 g of cells).The suspension was sonicated for 10 minutes (10 s pulse ON, 10 s pulse OFF, 45% amp) and kept at 0 °C.Next, the resulting suspension was centrifuged at 4 °C and 14,000 rpm for 60 min.Finally, the supernatant was filtered (0.45 µm filter) and collected for analysis.
Firstly, 40 mL solutions of E. coli cell lysate were freeze-dried.Then the residue was reconstituted with 4 mL of 20 mM AmAc.After centrifuging the obtained solution at 5 °C and 12,000 rpm for an hour, the supernatant was collected, after which it was centrifuged again at the same conditions.Next, the obtained clear supernatant was concentrated with spin filters (cut-off MW: 3 kDa) at 5 °C and 12,000 rpm for 2 to 3 hours.The acquired solution was stored at -20 °C.

Figure S2 .
Figure S2.Optimization of the concentration of salts (pH 8.5) for salt-mediated pH-gradient separations.(a) Chromatograms of protein mixtures.(b) Chromatograms of mixtures of mAbs.MPA = mobile phase A.

Figure
Figure S5.(a) EIC of BSA different concentrations (0.1 mg/mL to 1 mg/mL with 1 µL injection).The m/z values used in EIC are 3908.42,4152.76,4429.53,4745.81.(b) The plot of relationship between the µg injection of BSA and corresponding peak areas.

Figure S8 .
Figure S8.The analysis of protein complexes (asparaginase and ADH) with nanoSCX-nMS method.(a) and mass spectrum (b) of the asparaginase.Separation (c) and mass spectrum (d) of the ADH.Concentrations of asparaginase and ADH: 0.1 mg/mL.Injection: 1 µL.

Figure S9 .
Figure S9.The standard curve of BSA measured with the Bradford method.The concentration of the E. coli cell lysate was obtained by the equation of 0.61−0.007230.6374 × 10, namely 9.457 mg/mL.

Figure
Figure S11.(a) 2D plot of deconvolution results of the E. coli cell lysate with UniDec.Deconvolution parameters are reported in the data analysis section.(b and c) Average mass spectra and deconvoluted spectra of the high-MW species.

Table S1 .
Basic information on proteins and mAbs.

Table S3 .
Parameters for packing SCX columns and trap columns.